Previously, we have proposed that the beta 1 residues 9-13, 26, 28 and 86 in HLA-DR17, the most common subtype of DR3, might be critical for the binding of an immunodominant, mycobacterial epitope (peptide 3-13 of the 65-kDa heat shock protein). In order to examine directly (i) which DR17 residues are involved in peptide binding, (ii) whether the same or other DR17 residues are involved in the binding of different peptides, and (iii) whether subtle differences in the mode of peptide binding can influence T cell stimulation, we have now systematically mutated 15 highly polymorphic DR17 beta 1 residues, located in the proposed peptide binding groove of DR17, and examined the effect thereof on binding and presentation of two peptides, hsp65 p3-13 and p56-65 of the 30/31-kDa secreted mycobacterial protein. Mutations in residues 28 (D-->H) and 86 (V-->G) completely eliminated binding of p3-13 and significantly reduced binding of p56-65. A mutation in residue 26 (Y-->F) decreased binding of p3-13 but did not affect binding of p56-65. Substitutions of amino acid residues 28, 67, 71 and 86 in the DR17 beta 1 chain abrogated peptide-specific stimulation of both the p3-13- and the p56-65-specific T cell clones, while specific stimulation by only one peptide was eliminated by substitution at positions 26 and 74 (p3-13) and by substitution of residues 11 and 37 (p56-65). The observation that substitution of several other peptide-contacting DR17 beta 1 chain residues does not significantly affect peptide binding but does affect T cell stimulation, suggests that these substitutions alter the conformation of the bound peptide.